How Can Plants Tell Which Way Is Up? Laboratory Exercises to Introduce Gravitropisyn John

How Can Plants Tell Which Way Is Up? Laboratory Exercises To Introduce Gravitropisyn John Z. Kiss Sean E. Weise Helen G, Kiss Many people think of plants as The laboratory exercises described structures. In higher plants, a great deal essentially sessile organisms that do not in this paper involve the higher plant of evidence suggests that amyloplasts actively respond to their environment. Arabidopsis thaliana, which has become (starch-filled plastids), which settle or What could be further from the truth! an important organism in molecular In fact, plants are capable of a variety of sediment to the new lower cell wall biology research and is the focus of an following reorientation, function as movements, including the dramatic international plant genome project. nastic responses (such as Venus fly trap statoliths. Thus, the idea to explain Based on the material presented here, a gravity perception in plants has been closure) and the less sensational number of plant gravitropism labora- tropisms. These latter movements are referred to as the "starch statolith tory exercises with Arabidopsis that are hypothesis," and the evidence supporting directed growth responses to some type simple in terms of equipment/materi- of external stimulus such as gravity als and procedures can be developed. this hypothesis has been reviewed in a (gravitropism, formerly known as These exercises are robust in that they number of publications (e.g. Sack 1997), geotropism) or light (phototropism). work well even in the hands of intro- including a very accessible article in This paper describes some interesting ductory students, and they can be Scientific American (Evans et al. 1986). exercises that are derived from recent expanded according to the individual Some of the strongest evidence that work, including research that has led to instructor's needs. This paper describes amyloplasts function as statoliths experiments performed on two Space two exercises that have been per- comes from research with mutants of Shuttle missions in 1997 (Kiss et al. formed by beginning college students, Arabidopsis that lack starch (Kiss et al. 1998). and these exercises can easily be per- 1997). These mutants are starchless The study of tropisms can be a useful formed in biology classes in most high since they are missing one of the way to introduce students to plant school settings. biology in high school and introductory enzymes (phosphoglucomutase or college courses. In our experience, pgm) in the final steps of starch students are fascinated by plant move- synthesis. Several studies have shown ments when they are presented in lec- Background that the starchless mutants are much tures and find laboratory experiences less sensitive to gravity, but these on this topic quite engaging. Laboratory mutants still do respond to gravity (i.e. work on plant tropisms can also be Gravitropism in plants can be they are not agravitropic). The upshot used to introduce important concepts divided into three temporal stages: of this work is that plastids (with starch in science such as hypothesis testing, perception, transduction/transmission, in the case of the normal wild-type, quantitative analysis, and the use of and response (i.e. the differential WT, and without starch in the case of statistics. growth in a plant that leads to the mutant) can function in gravity downward or upward curvature). perception. (It is important to note that These laboratory exercises are focused both the WT and mutant plants have on the early events of plant the same number of plastids, but the gravitropism termed "gravity mutant has starchless plastids.) How- perception." Gravitropism and other ever, when starch is present, there is a John Z. Kiss is Professor of Botany at greater plastid density and an increased Miami University in Oxford, OH 45056; e- tropisms in plants have been exten- mail: [email protected], and has sively studied since late in the last total mass of these statoliths, and the a o experiments y on the Space Shuttle. century, and Charles Darwin and his plants do better in terms of gravity Sean E. Weise was an undergraduate son Francis published a book titled The perception and response. botany major at Miami University. Helen Power of Movements in Plants in 1883. In the two related laboratory exer- G. Kiss is in the Office for the cises that follow, students should be Advancement of Scholarship and Teaching (In fact, the Darwins' early experiments and serves as an adjunct faculty member in phototropism are outlined in several able to demonstrate that while both the in Botany at Miami University. introductory biology textbooks.) WT and mutant respond to gravity, the Gravity perception in plants is WT has a greater response. One hypothesized to be mediated by the exercise focuses on gravitropism in the interaction of dense organelles (called INTRODUCING GRAVITROPISM 59 flower stalks of mature plants, and the to provide a humid atmosphere to experiment, and if the plants are left in other involves a study of gravitropism in stimulate germination. At this point, the favorable conditions, they will continue young seedlings. They can be performed pots are placed under continuous to produce additional stalks that can be independently or together, depending on illumination from 40-watt fluorescent used for other experiments.) the instructor and his or her course. bulbs in a "shop" light fixture. Re- Curvature is measured at each time markably, the starchless mutant and WT interval with a protractor, and the plants are identical in morphology and size if then are returned to the box. Sample data they are maintained together under are shown in Table 1. Students can Exercise #I -Flower Stalks continuous illumination. If plants are observe that WT flower stalks curve from Mature Plants grown on a light/dark cycle, then the faster compared to the mutant flower mutant may be smaller and develop at a stalks, even though the growth rates of Materials Needed slower rate compared to the WT. both strains are similar. The data can be Seeds of normal WT Arabidopsis and a However, we have found that the growth presented in table format or values can starchless mutant are available free of rate, in continuous light, of the WT and be used to draw a graph. Instructors can charge (or in some cases, for a small fee) mutant flower stalks is almost identical; also have the students perform some to educators through the Arabidopsis thus potential differences in growth are simple statistical analyses (such as a Biological Resource Center (ABRC) at not significant when evaluating Mest) to determine if the curvature Ohio State University. Orders may be gravitropism. values are significantly different placed through the Internet An alternate to growing the plants in a between the WT and the mutant flower (http://aims.cps.msu.edu/aims/), by e- soil mixture is to use a synthetic stalks. mail ([email protected]), or by substrate, such as Rockwool cubes or If a light-tight box is not practical to standard mail (ABRC, 309 B & Z Oasis floral foam cubes, purchased from implement, it is possible to do this Building, 1735 Neil Ave., Columbus, a greenhouse or gardening supply store. experiment in general room light since OH 43210; telephone: 614-292-9371; The advantage of this method is that the phototropic response is much weaker fax: 614-292-0603). The starchless there is no potential problem with soil than the gravitropic response in these mutant is pgm-1 (stock no. CS210), and spilling when the plants are reoriented. flower stalks. Another variation is the WT strain to use is Columbia (Col- In any case, soil moisture should be simply to do the experiment with the 21stock number CS907). The ABRC will carefully maintained until the seeds WT only, and this exercise still allows not send many seeds, but you can grow germinate, which should be in about students to appreciate the phenomenon more plants for your own seed stock (i.e. three to five days after sowing. Under in plants and to do some data analysis. plants self-pollinate), and each plant will conditions of low humidity, it may take a The upward curvature of WT flower produce thousands of seeds. Other few more days for germination. Seeds stalks is illustrated in Figure 1. materials needed for this exercise will germinate at room temperature (20 This exercise can be enhanced by include soil, pots, a light bank to 22° C), but temperatures above 25° C demonstrating the differing phenotypes (fluorescent "shop" lights work well), a should be avoided. Following of the WT and starchless mutant. Leaves light-tight box (e.g. photocopy paper germination, plants should be watered as from the plants are excised and left boxes or large shoe boxes), and pro- needed, but too much watering may lead overnight in ethanol to remove the to fungal growth. Arabidopsis is in the chlorophyll. The leaves then are placed mustard family, and the plants will form in an iodine solution (i.e. 2.0 g potass- Preparation rosettes of leaves. Once the plants are at ium iodide and 0.2 g iodine are added to You will need to grow plants in pots the rosette stage, they may need to be 100 mL of water), and the WT will stain until they start forming flower stalks. thinned to avoid overcrowding in the intensely black while there will be no There are many ways to do this, but we pots. About three to four weeks after reaction in the starchless mutant. will provide highlights of one general sowing of seeds, the rosettes will start to Exercise #2-Young method here, and the reader is referred form flower stalks or inflorescences. The Seedlings to the above ABRC site to learn more gravitropism experiments work best on details (if needed) and alternatives in flower stalks that are 2 to 6 cm in length.

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